Lipid nanoparticles are the most clinically advanced delivery vehicles for nucleic acid-based therapeutics, as demonstrated by the mRNA-based vaccines against SARS-CoV-2 from Moderna and Pfizer/BioNTECH. Applications of mRNA delivery expand beyond vaccines to include genome editing, protein replacement therapy, and immunotherapy. These lipid nanoparticles function to protect mRNA against degradation during transit and facilitate endosomal escape for the efficient delivery of their mRNA cargo upon cell internalization. However, nanoparticles encounter various biological tissues and compartments upon injection and biomolecules such as proteins spontaneously adsorb to the nanoparticle surface, coating the lipid nanoparticles in a protein “coronaâ€. Currently, we do not understand the connection between the protein corona formed and the delivery outcomes observed for lipid nanoparticles. Moreover, most studies of protein corona formation to date have employed dense nanoparticles with rigid structures (such as metal- or polystyrene-based nanoparticles), yet we expect soft nanoparticles will be the standard for mRNA-based delivery systems. As such, a better understanding of these biomolecular interactions on the surface of lipid nanoparticles may be critical to move beyond conventional outcomes of intravenous injection resulting in liver localization or the requirement for local delivery (i.e., intramuscular injection).
In this work, we have developed a quantitative, label-free mass spectrometry-based proteomics workflow to probe the nano-bio interface of soft lipid nanoparticles. We provide clarity on the experimental challenges of separating protein-nanoparticle complexes from free proteins present in bulk biofluids. Using this method, we investigate how altering the (i) ionizable lipid within the lipid nanoparticle and (ii) mRNA cargo impact the formation of the protein corona in (iii) various relevant delivery bioenvironments. These techniques are readily translatable to study other soft nanoparticle-based systems. By elucidating these fundamental protein-nanoparticle interactions, we can rationally tune the design of these mRNA-based biotechnologies for improved translation to clinical practice.
